Long-chain dibasic acid with low content of monobasic acid impurity and the production method thereof

ABSTRACT

The invention relates to a long-chain dibasic acid with low content of monobasic acid impurity and a production method thereof, in particular to the preparation of a long-chain dibasic acid producing strain by means of directed evolution and homologous recombination, and to the production of a long-chain dibasic acid with low content of monobasic acid impurity by fermentation of said strain. The invention relates to a mutated CYP52A12 gene, homologous gene or variant thereof, which, relative to GenBank Accession Number AY230498 and taking the first base upstream of the start codon ATG as −1, comprises a mutation. The invention relates to a strain comprising said mutated CYP52A12 gene, homologous gene or variant thereof wherein when the strain is fermented to produce a long-chain dibasic acid, the content of monobasic acid impurity in the fermentation product is significantly reduced.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of Chinese PatentApplication No. 201810734273.7, filed on Jul. 6, 2018 and Chinese PatentApplication No. 201810734190.8 filed on Jul. 6, 2018, and Chinese PatentApplication No. 201910321614.2, filed on Apr. 22, 2019, the disclosuresof which are incorporated herein by reference in their entirety.

SEQUENCE STATEMENT

Incorporated by reference herein in its entirety is the Sequence Listingentitled “0503.13_ST25.txt”, size of 76 kilobytes, created Feb. 3, 2021.

FIELD OF THE INVENTION

The invention relates to a long-chain dibasic acid with low content ofmonobasic acid impurity and a preparation method thereof; as well as toa method for preparing a long-chain dibasic acid producing strain byusing directed evolution and homologous recombination, and a method forproducing a long-chain dibasic acid with low content of monobasic acidimpurity by using said strain.

BACKGROUND

Long-chain dibasic acid (LCDA; also referred to as long chaindicarboxylic acid or long chain diacid) is a dibasic acid comprising theformula HOOC(CH₂)_(n)COOH, where n≥7. As important monomer rawmaterials, long-chain dibasic acids are widely used in the synthesis ofnylon, resins, hot-melt adhesives, powder coatings, preservatives,perfumes, lubricants, plasticizers, and the like.

For a long time, long-chain dibasic acids are synthesized via classicalchemical synthesis pathway from the petroleum, such as butadienemultiple-step oxidation process. The Chemical synthesis methods facemany challenges. Dibasic acid obtained by the chemical synthesis methodis a mixture of long-chain dibasic acid and short-chain dibasic acid,and thus the subsequent complicated extraction and purification stepsare necessary, which become a huge obstacle for production technique andproduction cost. Microbiological fermentation technology of producing along-chain dibasic acid is advantageous over classical chemicalsynthesis method because it produces less pollution, is environmentfriendly, and is capable of synthesizing a long-chain dibasic acid suchas the one having more than 12 carbon atoms which is difficult to beproduced by chemical synthesis methods and has high purity and the like.

But using the microbiological fermentation method for producing along-chain dibasic acid, there are residual impurities in the productsometimes and the reduction in the product purity affects the quality ofthe product significantly, which greatly affects its later application.In particular, the impurity which is characteristically similar tolong-chain dibasic acid not only generates technical challenges to thelater extraction and purification, but also produces great negativeeffects on the production cost control. Therefore, to genetically modifya strain for producing a long-chain dibasic acid so as to reduce thecontent of certain impurity during fermentation is important andvaluable in industry to dibasic acid production via biologicalsynthesis.

Previously, the improvement of a dibasic acid producing strain wasmostly achieved through conventional random mutagenesis or geneticengineering methods. Due to the characteristic of random mutagenesis,there is a high requirement for screening throughput, and a new round ofmutagenesis screening is required for each changed trait, which hasbecome an important limiting factor technically. The genetic engineeringmeans can be used to perform targeted genetic modification of a strain,so as to obtain a good strain with higher yield. The microbiologicalfermentation method of a long-chain dibasic acid is mainly based onω-oxidation of alkane, which can then be degraded by β-oxidationpathway. Previous studies have shown that the yield of a long-chaindibasic acid can be increased by means of enhancing the ω-oxidationpathway and inhibiting the β-oxidation pathway. Pictaggio et al. ofCoginis company (Mol. Cell. Biol., 11(9), 4333-4339, 1991) reported thatknocking out two alleles of each of POX4 and POX5 could effectivelyblock the β-oxidation pathway to achieve 100% conversion rate of thesubstrate. Further overexpression of the genes of two key enzymes, P450and oxidoreductase CPR-b, of the rate-limiting step in the ω-oxidationpathway could effectively increase production. Xiaoqin Lai et al.(Chinese patent CN103992959B) reported that the introduction of one copyof the CYP52A14 gene into a dibasic acid producing strain could alsoeffectively increase the conversion rate and production efficiency ofthe dibasic acid. In addition, Zhu'an Cao et al. (Biotechnol. J., 1,68-74, 2006) from Tsinghua University found that knocking out a copy ofthe key gene CAT in the transportation of acetyl coenzyme A fromperoxisome to mitochondria could partially block acetyl coenzyme Aentering the citric acid cycle, and also effectively reduce thedegradation of dibasic acids.

Error-prone PCR is the technique proposed by Leung et al. (Technical, 1,11-15, 1989) to construct a gene library for directed studies. Bychanging PCR conditions, such as adjusting the concentration of fourdeoxyribonucleic acids in the reaction system, changing theconcentration of Mg²⁺, and using a low-fidelity DNA polymerase and thelike, the bases are mismatched so as to introduce a mutation. Too highor too low mutation rate will affect the effect of constructing mutantlibraries. The ideal base mutation ratio is 1-3 per DNA fragment.Therefore, the beneficial mutations that contribute to furtherimprovement of the strain productivity can be screened throughgene-directed genetic modification by using error-prone PCR ofgenerating random mutations in combination with homologousrecombination.

Previous studies on dibasic acid producing strains focused on randommutagenesis or overexpression of a gene in the upstream syntheticpathway or blocking the downstream β-oxidation pathway, and the directedevolution of a gene in the metabolic pathway has not been reported orapplied. There is still a need in the art for a strain thatsignificantly increases the yield of a long-chain dibasic acid andsignificantly reduces the content of some impurities, as well aspreparation methods thereof.

SUMMARY OF THE INVENTION

The invention relates to an isolated mutated CYP52A12 gene, homologousgene or variant thereof, which, relative to GenBank Accession NumberAY230498 (e.g. set forth in SEQ ID NO: 21, in particular nucleotides1-1176 or 265-1176) and taking the first base (e.g. the base “C” atposition 1176 of SEQ ID NO: 21) upstream of the start codon ATG (wherein“A” is position 1) as −1, comprises any one or more of the followingbase mutations: −876A>G; −853A>T; −831delT; −825C>A; −823delG; −579A>G;−412_−411AC>TT; −402insTT and −15_1 ACCAACCAACCAACCA (SEQ IDNO.:37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA), wherein thevariant has at least 70% sequence identity to the mutated CYP52A12 geneor homologous gene thereof.

In an embodiment, the mutated CYP52A12 gene, homologous gene or variantthereof comprises the base mutation −15_1 ACCAACCAACCAACCA (SEQ IDNO.:37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA), e.g. setforth in SEQ ID NO: 22.

In an embodiment, the mutated CYP52A12 gene, homologous gene or variantthereof comprises the base mutations −412_−411AC>TT; −402insTT and −15_1ACCAACCAACCAACCA (SEQ ID NO.:37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g.−7_−4delACCA), e.g. set forth in SEQ ID NO: 23.

In an embodiment, the mutated CYP52A12 gene, homologous gene or variantthereof comprises the base mutations −579A>G; −412_−411AC>TT; −402insTTand −15_1 ACCAACCAACCAACCA (SEQ ID NO.:37)>ACCAACCAACCA (SEQ ID NO.: 38)(e.g. −7_−4delACCA), e.g. set forth in SEQ ID NO: 24.

In an embodiment, the mutated CYP52A12 gene, homologous gene or variantthereof comprises the base mutations −831delT; −825C>A; −823delG;−579A>G; −412_−411AC>TT; −402insTT and −15_1 ACCAACCAACCAACCA (SEQ IDNO.:37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA), e.g. setforth in SEQ ID NO: 25.

In an embodiment, the mutated CYP52A12 gene, homologous gene or variantthereof comprises the base mutations −876A>G; −853A>T; −831delT;−825C>A; −823delG; −579A>G; −412_−411AC>TT; −402insTT and −15_1ACCAACCAACCAACCA (SEQ ID NO.:37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g.−7_−4delACCA), e.g. set forth in SEQ ID NO: 26.

The invention relates to an isolated DNA molecule, which, relative tonucleotides 1-1176 or 265-1176 of SEQ ID NO: 21, comprises any one ormore of the following base mutations 301A>G, 324A>T, 346delT, 352C>A,354delG, 598A>G, 765_766AC>TT, 774insTT and 1162_1176ACCAACCAACCAACC(SEQ ID NO.: 39)>ACCAACCAACC (SEQ ID NO.:40)(e.g. 1170_1173delACCA).

In an embodiment, the isolated DNA molecule comprises the base mutation1162_1176ACCAACCAACCAACC (SEQ ID NO.: 39)>ACCAACCAACC (SEQ ID NO.:40)(e.g. 1170_1173delACCA), e.g. set forth in SEQ ID NO: 27 or 32.

In an embodiment, the isolated DNA molecule comprises the base mutations765_766AC>TT, 774insTT and 1162_1176ACCAACCAACCAACC (SEQ ID NO.:39)>ACCAACCAACC (SEQ ID NO.:40) (e.g. 1170_1173delACCA), e.g. set forthin SEQ ID NO: 28 or 33.

In an embodiment, the isolated DNA molecule comprises the base mutations598A>G, 765_766AC>TT, 774insTT and 1162_1176ACCAACCAACCAACC (SEQ ID NO.:39)>ACCAACCAACC (SEQ ID NO.:40) (e.g. 1170_1173delACCA), e.g. set forthin SEQ ID NO: 29 or 34.

In an embodiment, the isolated DNA molecule comprises the base mutations346delT, 352C>A, 354delG, 598A>G, 765_766AC>TT, 774insTT and1162_1176ACCAACCAACCAACC (SEQ ID NO.: 39)>ACCAACCAACC (SEQ ID NO.:40)(e.g. 1170_1173delACCA), e.g. set forth in SEQ ID NO: 30 or 35.

In an embodiment, the isolated DNA molecule comprises the base mutations301A>G, 324A>T, 346delT, 352C>A, 354delG, 598A>G, 765_766AC>TT, 774insTTand 1162_1176ACCAACCAACCAACC(SEQ ID NO.: 39)>ACCAACCAACC (SEQ ID NO.:40)(e.g. 1170_1173delACCA), e.g. set forth in SEQ ID NO: 31 or 36.

In an embodiment, the sequence of the mutated CYP52A12 gene according tothe invention is set forth in any of SEQ ID NOs. 16 and 22-26 or asequence having at least 70% sequence identity thereto, for example atleast about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%,99.91%, 99.92%, 99.93%, 99.94%, 99.95%, or 99.96% sequence identity.

The invention further relates to a microorganism containing the isolatedDNA molecule (for controlling the expression of CYP52A12 gene, e.g. as apromoter) or the mutated CYP52A12 gene, homologous gene or variantthereof according to the invention, which produces a long-chain dibasicacid with a reduced content of monobasic acid impurity, compared to amicroorganism containing a non-mutated CYP52A12 gene or homologous genethereof.

The invention further relates to a method of producing a long-chaindibasic acid by fermentation with a microorganism containing theisolated DNA molecule or the mutated CYP52A12 gene, homologous gene orvariant thereof according to the invention, comprising a step ofculturing the microorganism, and optionally a step of isolating,extracting and/or purifying the long-chain dibasic acid from theculture.

In some embodiments, after the completion of the process of producing along-chain dibasic acid by fermentation with a microorganism accordingto the invention, the mass ratio of monobasic acid impurity infermentation broth is below 5%, wherein the mass ratio is the masspercentage of the monobasic acid impurity to the long-chain dibasic acidin the fermentation broth.

In some embodiments, after the completion of the process of producing along-chain dibasic acid by fermentation with a microorganism accordingto the invention, the content of monobasic acid impurity in thefermentation broth, compared with that of the monobasic acid impurity inthe long-chain dibasic acid produced by fermentation method with aconventional microorganism, such as by fermentation with a non-mutantmicroorganism according to the invention, is decreased by at least 5%.

The invention also relates to a long-chain dibasic acid with low contentof monobasic acid impurity, wherein the content of monobasic acidimpurity is greater than 0, and less than 12,000 ppm, preferably lessthan 10,000 ppm or 6,000 ppm, more preferably less than 3,000 ppm, morepreferably less than 1,000 ppm, more preferably less than 500 ppm, morepreferably less than 200 ppm; and wherein the monobasic acid impuritycomprises a long-chain monocarboxylic acid impurity, i.e. containingonly one carboxyl group (—COOH) in the carboxylic acid molecule.

In some embodiments, the long-chain dibasic acid producing microorganismstrain according to the invention contains the isolated DNA molecule(for controlling the expression of CYP52A12 gene, e.g. as a promoter) orthe mutated CYP52A12 gene, homologous gene or variant thereof accordingto the invention. In some embodiments, the long-chain dibasic acidproducing microorganism strain is the microorganism according to theinvention which contains the isolated DNA molecule or the mutatedCYP52A12 gene, homologous gene or variant thereof according to theinvention.

In some embodiments, the microorganism of the invention is selected fromthe group consisting of Corynebacterium, Geotrichum candidum, Candida,Pichia, Rhodotroula, Saccharomyces and Yarrowia, more preferably themicroorganism is yeast, and more preferably the microorganism is Candidatropicalis or Candida sake. In a particular embodiment, themicroorganism is CCTCC M2011192 or CCTCC M203052.

In some embodiments, the long-chain dibasic acid is selected from C9 toC22 long-chain dibasic acids, preferably selected from C9 to C18long-chain dibasic acids, more preferably one or more of C10 dibasicacid, C11 dibasic acid, C12 dibasic acid, C13 dibasic acid, C14 dibasicacid, C15 dibasic acid and C16 dibasic acid. More preferably, thelong-chain dibasic acid is at least one or more of C10 to C16 dibasicacids, or at least one or more of n-C10 to C16 dibasic acids, e.g. atleast one or more selected from the group consisting of sebacic acid,undecanedioic acid, dodecanedioic acid, tridecanedioic acid,tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid.

In some embodiments, the monobasic acid impurity according to theinvention has the formula of CH₃—(CH₂)n-COOH, where n≥7, and/orCH₂OH—(CH₂)n-COOH, where n≥7;

preferably the monobasic acid impurity comprises, but not limited to, along-chain monobasic acid with the number of carbon atoms in the carbonchain being greater than 9; preferably the monobasic acid impuritycomprises any one or more of a monobasic acid having 9 carbon atoms, amonobasic acid having 10 carbon atoms, a monobasic acid having 11 carbonatoms, a monobasic acid having 12 carbon atoms, a monobasic acid having13 carbon atoms, a monobasic acid having 14 carbon atoms, a monobasicacid having 15 carbon atoms, a monobasic acid having 16 carbon atoms, amonobasic acid having 17 carbon atoms, a monobasic acid having 18 carbonatoms, and a monobasic acid having 19 carbon atoms.

In some embodiments, where the long-chain dibasic acid is C12 dibasicacid e.g. dodecanedioic acid, the monobasic acid impurity ispredominantly a monobasic acid impurity having 12 carbon atoms, and thecontent of the monobasic acid impurity having 12 carbon atoms is lessthan 8,000 ppm.

In some embodiments, where the long-chain dibasic acid is C10 dibasicacid e.g. decanedioic acid, the monobasic acid impurity is predominantlya monobasic acid impurity having 10 carbon atoms, and the content of themonobasic acid impurity having 10 carbon atoms is less than 2,500 ppm.

In some embodiments, where the long-chain dibasic acid is C16 dibasicacid e.g. hexadecanedioic acid, the monobasic acid impurity ispredominantly a monobasic acid impurity having 16 carbon atoms, and thecontent of the monobasic acid impurity having 16 carbon atoms is lessthan 12,000 ppm.

The invention further relates to a method of modifying a long-chaindibasic acid producing microorganism strain, comprising a step ofdirected evolution of a key gene in the pathway of the long-chaindibasic acid synthesis, wherein, compared to the microorganism strainbefore modified, the modified long chain dibasic acid producingmicroorganism strain is capable of producing the long chain dibasic acidwith substantially decreased content of monobasic acid impurity, e.g.under the same conditions. In some embodiments, the key gene in thepathway of the long-chain dibasic acid synthesis is CYP52A12 gene.

In some embodiments, the microorganism of the invention is selected fromthe group consisting of Corynebacterium, Geotrichum candidum, Candida,Pichia, Rhodotroula, Saccharomyces and Yarrowia, more preferably themicroorganism is yeast, and more preferably the microorganism is Candidatropicalis or Candida sake. In a particular embodiment, themicroorganism is CCTCC M2011192 or CCTCC M203052.

In some embodiments, the long-chain dibasic acid according to theinvention is selected from C9 to C22 long-chain dibasic acids,preferably selected from C9 to C18 long-chain dibasic acids, morepreferably one or more selected from the group consisting of C10 dibasicacid, C11 dibasic acid, C12 dibasic acid, C13 dibasic acid, C14 dibasicacid, C15 dibasic acid and C16 dibasic acid. More preferably, thelong-chain dibasic acid is at least one or more of C10 to C16 dibasicacids, or at least one or more of n-C10 to C16 dibasic acids, e.g. atleast one or more selected from the group consisting of sebacic acid,undecanedioic acid, dodecanedioic acid, tridecanedioic acid,tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid.

In some embodiments, the content of monobasic acid impurity, comparedwith that of the monobasic acid impurity in the long-chain dibasic acidproduced by fermentation method with a conventional microorganism, isdecreased by at least 5%, preferably at least 10%, more preferably atleast 20%, more preferably at least 40%, and more preferably at least50% or more.

In some embodiments, the method of modifying a long-chain dibasic acidproducing microorganism strain comprises steps of:

1) preparing a target gene (CYP52A12 gene) fragment having a mutation byerror-prone PCR;

2) preparing fragments upstream and downstream of the target gene(CYP52A12 gene) necessary for homologous recombination as templates forhomologous recombination with a resistance marker gene, preferably theresistance marker gene is hygromycin B;

3) preparing a complete recombination fragment by PCR overlap extension;

4) introducing the recombination fragment into a strain by homologousrecombination;

5) screening positive strains by means of the resistance marker;

6) screening strains wherein the content of monobasic acid impurity inthe fermentation broth after completion of fermentation is significantlydecreased;

7) optionally, removing the resistance marker in the screened strains byfurther homologous recombination.

The invention further relates to a fermentation broth during a processof producing a long-chain dibasic acid by fermentation with amicroorganism, wherein the fermentation broth contains monobasic acidimpurity, and the mass ratio of monobasic acid impurity is less than 5%,preferably less than 1.5%, more preferably less than 1.0%, less than0.9%, wherein the mass ratio is the mass percentage of monobasic acidimpurity to the long-chain dibasic acid in the fermentation broth.

Preferably, the long-chain dibasic acid is selected from C9 to C22long-chain dibasic acids, and the monobasic acid impurity comprises, butnot limited to, a long-chain monobasic acid with the number of carbonatoms in the carbon chain being greater than 9.

In some embodiments, the microorganism contains the isolated DNAmolecule (for controlling the expression of CYP52A12 gene, e.g. as apromoter) or the mutated CYP52A12 gene, homologous gene or variantthereof according to the invention. In some embodiments, themicroorganism is the microorganism according to the invention whichcontains the isolated DNA molecule or the mutated CYP52A12 gene,homologous gene or variant thereof according to the invention. In someembodiments, the fermentation broth is obtained by a method of producinga long-chain dibasic acid by fermentation with a microorganismcontaining the isolated DNA molecule or the mutated CYP52A12 gene,homologous gene or variant thereof according to the invention. In someembodiments, the fermentation broth is obtained in a process ofproducing a long-chain dibasic acid with a microorganism obtained by themethod of modifying a long-chain dibasic acid producing microorganismstrain according to the invention.

The invention further relates to a method of producing a long-chaindibasic acid as described in the invention, comprising obtaining along-chain dibasic acid producing microorganism strain containing amutated CYP52A12 gene, a homologous gene or a variant thereof bydirected evolution of the CYP52A12 gene in the long-chain dibasic acidsynthesis pathway; culturing the strain to produce the long-chaindibasic acid by fermentation; optionally, further comprising the stepsof isolating, extracting and/or purifying the long-chain dibasic acidfrom the culture product;

wherein, the mutated CYP52A12 gene, homologous gene or variant thereof,which, relative to GenBank Accession Number AY230498 and taking thefirst base upstream of the start codon ATG (in which the “A” is 1) as−1, comprises any one or a combination of some of the following basemutations occurred in the promoter region thereof: −876A>G; −853A>T;−831delT; −825C>A; −823delG; −579A>G; −412_−411AC>TT; −402insTT; −15_1ACCAACCAACCAACCA (SEQ ID NO.: 37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g.−7_−4delACCA); and the variant has at least 70% sequence identity to themutated CYP52A12 gene or homologous gene thereof.

Preferably, the mutated CYP52A12 gene, homologous gene or variantthereof comprises the base mutation −15_1 ACCAACCAACCAACCA (SEQ ID NO.:37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA) in the promoterregion thereof.

Preferably, the mutated CYP52A12 gene, homologous gene or variantthereof comprises the base mutations −412_−411AC>TT; −402insTT and −15_1ACCAACCAACCAACCA (SEQ ID NO.: 37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g.−7_−4delACCA) in the promoter region thereof.

Preferably, the mutated CYP52A12 gene, homologous gene or variantthereof comprises the base mutations −579A>G; −412_−411AC>TT; −402insTTand −15_1 ACCAACCAACCAACCA (SEQ ID NO.: 37)>ACCAACCAACCA (SEQ ID NO.:38) (e.g. −7_−4delACCA) in the promoter region thereof.

Preferably, the mutated CYP52A12 gene, homologous gene or variantthereof comprises the base mutations −831delT; −825C>A; −823delG;−579A>G; −412_−411AC>TT; −402insTT and −15_1 ACCAACCAACCAACCA (SEQ IDNO.: 37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA) in thepromoter region thereof.

Preferably, the sequence of the mutated CYP52A12 gene is set forth inany of SEQ ID NOs. 16 and 22-26 or a sequence having at least 70%sequence identity thereto, for example at least about 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%,99.94%, 99.95%, or 99.96% sequence identity.

Preferably, the long-chain dibasic acid is selected from C9-C22long-chain dibasic acids, preferably selected from C9-C18 long-chaindibasic acids, more preferably one or more selected from the groupconsisting of C10 dibasic acid, C11 dibasic acid, C12 dibasic acid, C13dibasic acid, C14 dibasic acid, C15 dibasic acid and C16 dibasic acid.More preferably, the long-chain dibasic acid is at least one of C10 toC16 dibasic acids, or at least one of n-C10 to C16 dibasic acids, e.g.at least one selected from the group consisting of sebacic acid,undecanedioic acid, dodecanedioic acid, tridecanedioic acid,tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid.

Preferably, the chemical formula of the monobasic acid impuritycomprises CH₃—(CH₂)n-COOH, where n≥7, and/or CH₂OH—(CH₂)n-COOH, wheren≥7.

In some embodiments, the microorganism is yeast; more preferably themicroorganism is selected from Candida tropicalis or Candida sake.

In some embodiments, to obtain a long-chain dibasic acid producingmicroorganism strain with a mutated CYP52A12 gene, a homologous gene ora variant thereof, comprises the following steps:

1) preparing a CYP52A12 gene fragment having a mutation by error-pronePCR;

2) preparing fragments upstream and downstream of the CYP52A12 genenecessary for homologous recombination as templates for homologousrecombination with a resistance marker gene, preferably the resistancemarker gene is hygromycin B;

3) preparing a complete recombination fragment by PCR overlap extension;

4) introducing the recombination fragment into a strain by homologousrecombination;

5) screening positive strains by means of the resistance marker;

6) screening strains wherein the content of monobasic acid impurity inthe fermentation broth after fermentation is significantly reduced;

7) optionally, removing the resistance marker in the screened strains byfurther homologous recombination.

Preferably, in the present invention, the existing Candida tropicalisstrain CATN145 (Deposit No. CCTCC M2011192) is used as the startingstrain, hereinafter referred to as CCTCC M2011192, and error-prone PCRmethod is used to randomly mutate the CYP52A12 gene. The gene issubjected to directed evolution by homologous recombination method toscreen for a long-chain dibasic acid producing strain with significantlyreduced content of impurity during the production of dibasic acid,wherein the impurity is monobasic acid impurity.

By screening, the present invention obtains a strain in which thecontent of monobasic acid impurity in the fermentation product issignificantly reduced, and is named as mutant strain 430. By sequencinganalysis, it is found that, compared to parental strain CCTCC M2011192and taking the first base upstream of the start codon ATG (wherein “A”is 1) as −1, following base mutations occurred in the promoter region ofthe CYP52A12 gene of the mutant strain of Candida tropicalis screened inthe invention: −876A>G, −853A>T, −831delT, −825C>A, −823delG, −579A>G,−412_−411AC>TT, −402insTT, and −15_1 ACCAACCAACCAACCA (SEQ ID NO.:37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA). A promotersequence containing different combinations of mutation sites wassynthesized by the method of whole-genome synthesis (Sangon) (GenbankAccession No. AY230498), comprising 912 bp upstream of the start codonand adjacent 23 bp CDS sequence. Taking the first base upstream of thestart codon ATG as −1, any one or combination of several of the abovebase mutation sites can also achieve the effect of significantlydecreased content of monobasic acid impurity.

According to the present invention, the sequence of the Candidatropicalis CYP52A12 gene is set forth in SEQ ID NO: 16.

After further removing the resistance marker from the mutant strain,compared to the original strain, the mass ratio of monobasic acidimpurity in the fermentation broth after fermentation is significantlyreduced, and the content of monobasic acid impurity in the long-chaindibasic acid product obtained after extracting and purifying thefermentation broth can be reduced to 200 ppm or less, particularlyduring fermentation production of long-chain dodecanedioic acid.

The present invention screens a strain which has base mutations in thepromoter region of the CYP52A12 gene by directed evolution of said gene,and the content of monobasic acid impurity in the fermentation broth issignificant reduced for different fermentation substrates. Afterextraction and purification, compared to the parental strain, thecontent of monobasic acid impurity is decreased by nearly 50%, furtherimproving the purity of the fermentation product long-chain dibasicacid, making the dibasic acid product which is used as important rawmaterials for nylon filament, engineering plastic, synthetic fragrance,cold-resistant plasticizer, advanced lubricants and polyamide hot meltadhesives, favorable for the production of and the quality improvementof downstream products. More importantly, this greatly reduces thedifficulty of the extraction and purification processes at later stagesof dibasic acid production, simplifies the process and saves energy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a scheme of the integration of the CYP52A12 gene withmutations and the removal of the hygromycin resistance marker byhomologous recombination. “*” indicates the mutations that may bepresent in any region of CYP52A12 (including the promoter, codingregion, and terminator).

FIG. 2 is the alignment of the nucleotide sequences of the CYP52A12genes of the mutant strain of the invention (SEQ ID NO: 16) and theoriginal strain (nucleotides 265-1176 of SEQ ID NO: 21), and themutation sites are boxed with a black box, wherein 192 refers to CCTCCM2011192.

DETAILED DESCRIPTION Definition

Long chain alkane: the fermentation substrate of the invention comprisesa long chain alkane, belonging to a saturated aliphatic hydrocarbon,which is a saturated hydrocarbon among hydrocarbons; its whole structureis mostly composed only of carbon, hydrogen, carbon-carbon single bond,and carbon-hydrogen single bond. It includes an alkane of the formulaCH₃(CH₂)nCH₃, where n≥7. Preferred are n-C9-C22 alkanes, more preferredare n-C9-C18 alkanes, and most preferred are n-C10, C11, C12, C13, C14,C15 or C16 alkanes.

Long-chain dibasic acid (LCDA; also known as long chain dicarboxylicacid or long chain diacid, hereinafter abbreviated as dibasic acidsometimes) includes a diacid of the formula HOOC(CH₂)nCOOH, where n≥7.Preferably, the long-chain dibasic acid selected from C9-C22 long-chaindibasic acids, preferably selected from C9-C18 long-chain dibasic acids;more preferably comprises one or more of C10 dibasic acid, C11 dibasicacid, C12 dibasic acid, C13 dibasic acid, C14 dibasic acid, C15 dibasicacid and C16 dibasic acid. More preferably, the long-chain dibasic acidis at least one or more of C10 to C16 dibasic acids, preferably at leastone or more of n-C10 to C16 dibasic acids, e.g. at least one or moreselected from the group consisting of sebacic acid, undecanedioic acid,dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid,pentadecanedioic acid and hexadecanedioic acid.

Long-chain dibasic acid producing microorganism: the strain that hasbeen reported to produce and accumulate a dibasic acid includesbacterium, yeast, and mold, such as Corynebacterium, Geotrichumcandidum, Candida, Pichia, Rhodotroula, Saccharomyces, Yarrowia, and thelike. Among them, many species of Candida are good strains for theproduction of a dibasic acid by fermentation. The strain forfermentation preferably includes: Candida tropicalis or Candida sake.

In the process of producing a long-chain dibasic acid by fermentation ofa long-chain alkane substrate, alkane is oxidized to carboxylic acidcontaining only one carboxyl group. If the fermentation reaction is notcomplete, carboxylic acid with only one carboxyl group remains in thefermentation broth as impurity. Because of its extremely similarproperties to the long-chain dibasic acid, it is difficult to isolateefficiently by conventional means. Such impurity will enter the finaldibasic acid product along with the subsequent treatment process,greatly affecting the purity and quality of the product.

The monobasic acid impurity of the invention refers particularly to along-chain monocarboxylic acid impurity, which contains only onecarboxyl group (—COOH), particularly terminal carboxyl group, in thecarboxylic acid molecule. Preferably, the monobasic acid impuritycomprises, but not limited to, a long-chain monobasic acid with thenumber of carbon atoms in the carbon chain being greater than 9, whereinthe chemical formula of the monobasic acid impurity comprisesCH₃—(CH₂)n-COOH, where n≥7, and/or CH₂OH—(CH₂)n-COOH, where n≥7.

Preferably, the monobasic acid impurity is any one or more of amonobasic acid having 9 carbon atoms, a monobasic acid having 10 carbonatoms, a monobasic acid having 11 carbon atoms, a monobasic acid having12 carbon atoms, a monobasic acid having 13 carbon atoms, a monobasicacid having 14 carbon atoms, a monobasic acid having 15 carbon atoms, amonobasic acid having 16 carbon atoms, a monobasic acid having 17 carbonatoms, a monobasic acid having 18 carbon atoms, and a monobasic acidhaving 19 carbon atoms. The long-chain monobasic acid refers to a linearmonobasic acid containing one terminal carboxyl group, e.g. a monobasicacid having 9 carbon atoms refers to a long-chain monobasic acidcontaining 9 carbon atoms and having one terminal carboxyl group.

As used herein, the expression “substantially or significantly decreasedcontent of monobasic acid impurity” refers to that, compared to areference, the content of the fatty acid impurity (e.g. the totalcontent of monobasic acids CH₃—(CH₂)n-COOH and CH₂OH—(CH₂)n-COOH) isdecreased by at least 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%,25%, 30%, 35% 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, preferably atleast 10%, more preferably at least 20%, more preferably at least 40%,more preferably at least 50%, more preferably at least 70% or more.

When a long-chain dibasic acid is produced by fermentation according tothe present invention, the fermentation broth after fermentationcontains a monobasic acid impurity, and the content of the monobasicacid impurity is significantly reduced relative to the content of themonobasic acid impurity produced by a conventional microbiologicalfermentation, such as the fermentation by a non-mutant microorganismdescribed in the present invention, such as by at least 5%, 6%, 7%, 8%,9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%,80%, 90%, 95% or more, preferably at least 10%, more preferably at least20%, more preferably at least 40%, more preferably at least 50%, morepreferably at least 70% or more.

In some embodiments, the long-chain dibasic acid is produced by amicrobiological fermentation, and the fermentation broth contains amonobasic acid impurity, and the content of the monobasic acid impurityis reduced to below 5.0%, such as below 4.0%, 3.0%, 2.0%, 1.5%, 1.4%,1.3%, 1.2%, 1.1%, 1.0.%, 0.9%, 0.8%, 0.7%, 0.6% or lower, wherein themass ratio is a mass percentage of the monobasic acid impurity to thelong-chain dibasic acid in the fermentation broth, preferably reduced tobelow 1.5%, more preferably reduced to below 1.0%, and more preferablyreduced to below 0.9%.

The long-chain dibasic acid produced by the microbiological fermentationmethod of the present invention contains a monobasic acid impurity, andthe content of the monobasic acid impurity is greater than 0 and lessthan 15,000 ppm, preferably less than 10,000 ppm, less than 7,000 ppm,less than 5,000 ppm, less than 3,000 ppm, less than 2,000 ppm, less than1,000 ppm, less than 500 ppm, less than 300 ppm, less than 250 ppm, lessthan 200 ppm, less than 150 ppm, less than 100 ppm or less.

The unit ppm of the impurity content of the present invention is themass ratio of the impurity to the long-chain dibasic acid, and 100ppm=100*10⁻⁶=0.01%.

In some embodiments, the impurity of DC16 is higher than that of DC12 orDC10 on the whole, such as by at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, 60%, at least 80%, at least 100% or higher,wherein DC refers to long-chain dibasic acid.

In an embodiment of the present invention, when the C12 dibasic acidsuch as dodecanedioic acid is produced by the microbiologicalfermentation method of the invention, the monobasic acid impurity ispredominantly a monobasic acid impurity having 12 carbon atoms, and thecontent of the monobasic acid impurity having 12 carbon atoms is lessthan 8,000 ppm, such as less than 6,000 ppm, 5,000 ppm, 4,000 ppm, 3,000ppm, preferably less than 2,000 ppm, 1,900 ppm, 1,850 ppm, 1,800 ppm,1,750 ppm, 1,700 ppm, 1,650 ppm, 1,600 ppm, 1,550 ppm, 1,500 ppm, 1,450ppm, 1,400 ppm, 1,350 ppm, 1,300 ppm, 1,250 ppm, 1,200 ppm, 1,150 ppm,1,100 ppm, 1,050 ppm, 1,000 ppm, 950 ppm, 900 ppm, 850 ppm, 800 ppm, 750ppm, 700 ppm, 650 ppm, 600 ppm, 550 ppm, 500 ppm, 450 ppm, 400 ppm, 350ppm, 300 ppm, 250 ppm, 200 ppm, 150 ppm, 100 ppm, or less. The chemicalformula of the monobasic acid impurity having 12 carbon atoms comprisesCH₃—(CH₂)₁₀—COOH and/or CH₂OH—(CH₂)₁₀—COOH.

In an embodiment of the present invention, when the C10 dibasic acidsuch as decanedioic acid is produced by the microbiological fermentationmethod of the invention, the monobasic acid impurity is predominantly amonobasic acid impurity having 10 carbon atoms, and the content of themonobasic acid impurity having 10 carbon atoms is less than 2,000 ppm,such as less than 2,000 ppm, 1,500 ppm, preferably less than 1,000 ppm,500 ppm, 300 ppm, 250 ppm, 200 ppm, 150 ppm or less. The chemicalformula of the monobasic acid impurity having 10 carbon atoms comprisesCH₃—(CH₂)₈—COOH and/or CH₂OH—(CH₂)₈—COOH.

In an embodiment of the present invention, when the C16 dibasic acidsuch as hexadecanedioic acid is produced by the microbiologicalfermentation method of the invention, the monobasic acid impurity ispredominantly a monobasic acid impurity having 16 carbon atoms, and thecontent of the monobasic acid impurity having 16 carbon atoms is lessthan 12,000 ppm, such as less than 10,000 ppm, 9,000 ppm, 8,000 ppm,7,000 ppm, preferably less than 6,000 ppm, or less than 4,000 ppm, orless than 3,500 ppm, 3,000 ppm, 2,000 ppm, 1,000 ppm, 900 ppm, 800 ppm,700 ppm, 600 ppm, 500 ppm, 400 ppm, 300 ppm, 200 ppm, 150 ppm, or less.The chemical formula of the monobasic acid impurity having 16 carbonatoms comprises CH₃—(CH₂)₁₄—COOH and/or CH₂OH—(CH₂)₁₄—COOH.

In some embodiments, the content of monobasic acid impurity according tothe invention refers to the total content of monobasic acidsCH₃—(CH₂)n-COOH and CH₂OH—(CH₂)n-COOH.

The test method for the dibasic acid and the impurity content may employthe techniques well known to those skilled in the art, such as aninternal standard method or a normalization method of gas chromatographydetection.

CYP52A12 refers to one of the cytochrome oxidase P450 family CYP52subfamily, which forms a complex with cytochrome reductase CPR andparticipates in the ω-oxidation of alkanes and lipids duringfermentation production of acid. It is known to those skilled in the artthat the CYP52A12 gene or homologous gene thereof is also present inother microorganisms producing a long-chain dibasic acid, and theirsequences may differ, but are also within the scope of the presentinvention.

The term “isolated”, when applied to a nucleic acid or protein, meansthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It can be,for example, in a homogeneous state and may be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography.

As used herein, the expression “relative to the GenBank Accession NumberAY230498” or “relative to the nucleotide sequence of SEQ ID NO: 21”refers to a mutation at a corresponding position when aligned with thesequence of AY230498 or SEQ ID NO: 21. The corresponding position refersto the numbering of the residue of the reference sequence (SEQ ID NO:21) when the given polynucleotide sequence (e.g. a mutated CYP52A12 genesequence) is compared to the reference sequence. A base in a nucleicacid “corresponds” to a given base when it occupies the same essentialstructural position within the nucleic acid as the given base. Ingeneral, to identify corresponding positions, the sequences of nucleicacids are aligned so that the highest order match is obtained (see, e.g.Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;Carillo et al. (1988) SIAM J Applied Math 48: 1073). Alignment ofnucleotide sequences also can take into account conservative differencesand/or frequent substitutions in nucleotides. Conservative differencesare those that preserve the physico-chemical properties of the residuesinvolved. Alignments can be global (alignment of the compared sequencesover the entire length of the sequences and including all residues) orlocal (alignment of a portion of the sequences that includes only themost similar region(s)).

As used herein, the base mutation “XXX N0>N1” means that the base NO atposition XXX is mutated to the base N1; “XXXdelN” means that the base Nat position XXX is deleted; “XXXinasN0N1” means insertion of N0N1 afterposition XXX; “XXX0_XXX1 N0N1>N2N3” means the bases N0N1 at positionsXXX0 to XXX1 are mutated to N2N3; and “XXX0_XXX1 delN0N1N2N3” means thebases N0N1N2N3 at portions XXX0 to XXX1 are deleted.

For example, taking the first base upstream of the start codon ATG(wherein “A” is position 1) as −1, i.e. the first base immediatelyadjacent to the based “A” of the start codon “ATG” as −1, base mutation−876A>G means the base “A” at position −976 is mutated to “G”,“−831delT” means the base “T” at position −831 is deleted,“−412_−411AC>TT” means the bases “AC” at positions −412 and −411 aremutated to “TT”, −402insTT means bases “TT” are inserted after position−402 (i.e. between positions −402 and −403), and “−15_1 ACCAACCAACCAACCA(SEQ ID NO.: 37)>ACCAACCAACCA” (SEQ ID NO.: 38) means the bases“ACCAACCAACCAACCA” (SEQ ID NO.: 37) at positions −15 to 1 are mutated to“ACCAACCAACCA (SEQ ID NO.: 38).

In an embodiment, the sequence of the CYP52A12 gene according to theinvention is set forth in SEQ ID NO: 21, wherein the protein codingsequences are the nucleotides 1177 to 2748. Correspondingly, themutation “−876A>G” correspond to that the nucleotide “A” at position 301of SEQ ID NO: 21 is mutated to “G”; the mutation “−831delT” correspondto that the nucleotide “T” at position 346 of SEQ ID NO: 21 is deleted;the mutation “−412_−411AC>TT” correspond to that the nucleotides “AC” atpositions 765 and 766 of SEQ ID NO: 21 is mutated to “TT”; the mutation“−402insTT” correspond to the insertion of nucleotides “TT” between thepositions 774 and 775 of SEQ ID NO: 21; and the mutation “−15_1ACCAACCAACCAACCA (SEQ ID NO.: 37)>ACCAACCAACCA (SEQ ID NO.: 38)”correspond to that the nucleotides “ACCAACCAACCAACCA” (SEQ ID NO.: 37)at positions 1162-1177 of SEQ ID NO: 21 is mutated to “ACCAACCAACCA (SEQID NO.: 38) Herein, where a base is mentioned, G refers to guanine, Trefers to thymine, A refers to adenine, C refers to cytosine, and Urefers to uracil.

As used herein, the “non-mutated CYP52A12 gene” refers to a CYP52A12gene that does not comprises the mutation −876A>G; −853A>T; −831delT;−825C>A; −823delG; −579A>G; −412_−411AC>TT; −402insTT or −15_1ACCAACCAACCAACCA (SEQ ID NO.: 37)>ACCAACCAACCA (SEQ ID NO.: 38 (e.g.−7_−4delACCA) according to the invention, e.g. a naturally occurringwild type allele, such as the CYP52A12 gene with the Accession NumberAY230498 in the GenBank. An example of non-mutated CYP52A12 gene is setforth in SEQ ID NO: 21. The non-mutated CYP52A12 gene may contain othermutations, such as a silent mutation in the coding region which does notresult in the alteration of the encoded amino acid.

As used herein, “non-mutant microorganism” refers to a microorganismwhich does not contain the mutated CYP52A12 gene or homologous genethereof according to the invention, e.g. contain only the CYP52A12 genewith the Accession Number AY230498 in the GenBank. In an embodiment, thenon-mutant microorganism contains a non-mutated CYP52A12 gene accordingto the invention.

Using the method of the present invention, the present invention screensa strain having a mutated CYP52A12 gene, wherein, taking the first baseupstream of the start codon ATG (wherein the “A” is portion 1) as −1,any one or a combination of several of the following base mutationsoccurred in its promoter region: −876A>G; −853A>T; −831delT; −825C>A;−823delG; −579A>G; −412_−411AC>TT; −402insTT; −15_1 ACCAACCAACCAACCA(SEQ ID NO.: 37)>ACCAACCAACCA (SEQ ID NO.: 38) (e.g. −7_−4delACCA).

Homologous genes refer to two or more gene sequences with at least 80%similarity, including orthologous genes, paralogous genes and/orxenologous genes. The homologous gene of the CYP52A12 gene in theinvention refers to either the orthologous gene of the CYP52A12 gene, orparalogous gene or xenologous gene of the CYP52A12 gene.

Sequence identity refers to the percent identity of the residues of apolynucleotide sequence variant with a non-variant sequence aftersequence alignment and introduction of gaps. In some embodiments, thepolynucleotide variant has at least about 70%, at least about 75%, atleast about 80%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, atleast about 99.1%, at least about 99.2%, at least about 99.3%, 99.4%, atleast about 99.5%, at least about 99.6%, 99.7%, at least about 99.8%, atleast about 99.9%, at least about 99.91%, at least about 99.92%, atleast about 99.93%, at least about 99.94%, at least about 99.95%, or atleast about 99.96% polynucleotide or polypeptide homology with thepolynucleotide described herein.

As used herein, the terms “homology” and “identity” are usedinterchangeably herein to refer to the extent of non-variance ofnucleotide sequences, which can be detected through the number ofidentical nucleotide bases by aligning a polynucleotide with a referencepolynucleotide. The sequence identity can be determined by standardalignment algorithm programs used with default gap penalties establishedby each supplier. Homologous nucleic acid molecules refer to apre-determined number of identical or homologous nucleotides. Homologyincludes substitutions that do not change the encoded amino acid(“silent substitution”) as well as identical residues. Substantiallyhomologous nucleic acid molecules hybridize typically at moderatestringency or high stringency all along the length of the nucleic acidor along at least about 70%, 80% or 90% of the full-length nucleic acidmolecule of interest. Nucleic acid molecules that contain degeneratecodons in place of codons in the hybridizing nucleic acid molecule arealso contemplated in the invention. Whether any two nucleic acidmolecules have nucleotide sequences that are at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% “identical” can be determined using a knowncomputer algorithm such as the BLASTN, FASTA, DNAStar and Gap(University of Wisconsin Genetics Computer Group (UWG), Madison Wis.,USA). Percent homology or identity of nucleic acid molecules can bedetermined, e.g. by comparing sequence information using a GAP computerprogram (e.g., Needleman et al. J. Mol. Biol. 48: 443 (1970), as revisedby Smith and Waterman (Adv. Appl. Math. 2: 482 (1981)). Briefly, a GAPprogram defines similarity as the number of aligned symbols (i.e.,nucleotides) which are similar, divided by the total number of symbolsin the shorter of the two sequences.

Directed evolution refers to a process of simulating a natural selectionby technology means. Through an artificially created mutation andspecific screening pressure, a protein or nucleic acid is mutated in aspecific direction, thereby realizing an evolutionary process in naturethat requires thousands of years to complete in a short period of timeat the molecular level. The methods for performing directed evolutionare known in the art, e.g. error-prone PCR (e.g. Technique, 1, 11-15,1989; Genome Research, 2, 28-33, 1992).

In some embodiments, in the error-prone PCR of the invention, theconcentration of Mg²⁺ is in a range of 1 to 10 mM, preferably 2 to 8 mM,more preferably 5 to 6 mM, and/or the concentration of dNTP is from 0.1to 5 mM, preferably from 0.2 to 3 mM, more preferably 0.5 to 2 mM, andmore preferably from 0.8 to 1.5 mM, for example 1 mM, and/or addition offreshly prepared MnCl₂ to a final concentration of 0.1 to 5 mM,preferably 0.2 to 2 mM, more preferably 0.3 to 1 mM, and more preferably0.4 to 0.7 mM, such as 0.5 mM. In some embodiments, the rate of mutationis increased by decreasing the amount of template and appropriatelyincreasing PCR cycles to 40 or more, e.g. 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 55, 60 or more.

PCR overlap extension, also known as SOE (gene splicing by overlapextension) PCR, refers to a method of splicing different DNA fragmentstogether via PCR amplification by designing primers having complementaryends.

Homologous recombination refers to the recombination between DNAmolecules that relies on sequence similarity, most commonly found withincells to repair mutations that occur during mitosis. Homologousrecombination technology has been widely used in genome editing,including gene knockout, gene repair and introduction of a new gene to aspecific site. A class of microorganisms represented by Saccharomycescerevisiae has a very high rate of homologous recombination within cellswhich does not depend on sequence specificity and is obviouslyadvantageous in genome editing. Site-specific recombination relies onthe participation of specific sites and site-specific recombinases, andthe recombination occurs only between specific sites, such as Cre/loxP,FLP/FRT, and the like. The homologous recombination technology used inthe invention does not belong to site-specific recombination, andrecombination relies on the intracellular DNA repair system.

The resistance marker refers to a type of selective markers that oftenhas the ability of conferring a transformant survival in the presence ofan antibiotic. The resistance marker gene includes NPT, HYG, BLA andCAT, etc., which are resistant to kanamycin, hygromycin,ampicillin/carbenicillin, and chloramphenicol, respectively. Preferably,the resistance marker gene is the hygromycin B resistance gene HYG.

During fermentation, the fermentation medium comprises a carbon source,a nitrogen source, an inorganic salt and a nutritional factor.

In some embodiments, the carbon source comprises one or more selectedfrom the group consisting of glucose, sucrose and maltose; and/or thecarbon source is added in an amount of 1% to 10% (w/v), such as 1.5%,2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 6.0%, 7.0%, 8.0%, or 9.0%.

In some embodiments, the nitrogen source comprises one or more selectedfrom the group consisting of peptone, yeast extract, corn syrup,ammonium sulfate, urea, and potassium nitrate; and/or the nitrogensource is added in a total amount of 0.1%-3% (w/v), such as 0.2%, 0.4%,0.5%, 0.6%, 0.8%, 1.0%, 1.2%, 1.5%, 1.8%, 2.0%, or 2.5%.

In some embodiments, the inorganic salt comprises one or more selectedfrom the group consisting of potassium dihydrogen phosphate, potassiumchloride, magnesium sulfate, calcium chloride, ferric chloride, andcopper sulfate; and/or the inorganic salt is added in a total amount of0.1%-1.5% (w/v), such as 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%,1.0%, 1.1%, 1.2%, 1.3%, or 1.4%.

In some embodiments, the nutritional factor comprises one or moreselected from the group consisting of vitamin B1, vitamin B2, vitamin C,and biotin; and/or the nutritional factor is added in a total amount of0-1% (w/v), such as 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, or 0.9%.According to common knowledge in the art of fermentation, the percentagein the invention is the mass to volume ratio, i.e. w/v; and % indicatesg/100 mL.

Those skilled in the art can easily determine the amount of the abovesubstances to be added.

In one embodiment of the invention, the inoculation amount of the strainfor fermentation is 10% to 30%, for example 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 22%, 24%, 25%, 27%, or 29%. When the strain iscultured to an optical density (OD₆₂₀) of 0.5 or more (diluted 30folds), the substrate is added for fermentation conversion.

Extraction and purification of a long-chain dibasic acid: thefermentation broth containing a salt of a long-chain dibasic acidobtained by fermentation is subjected to extraction and purificationtreatment to obtain a long-chain dibasic acid product. The step ofextraction and purification comprises: sterilization of fermentationbroth, and acidification, solid-liquid separation, and/or solventcrystallization of the obtained clear solution. Some of fermentationbroth may contain a salt of a long-chain dibasic acid.

The extraction and purification according to the present invention maybe repeated more than once, and performing multiple extraction andpurification steps contribute to further reducing the impurity contentin the dibasic acid product. For example, in one embodiment of thepresent invention, with reference to the refining process in Example 1of Chinese Patent Application CN 101985416 A, the long-chaindodecanedioic acid product obtained by the present invention is furthertreated, and the content of the monobasic acid impurity having 12 carbonatoms in the obtained long-chain dodecanedioic acid can be reduced fromgreater than 5000 ppm before treatment to less than 4,000 ppm, forexample, less than 3000 ppm, less than 2000 ppm, less than 1000 ppm,less than 500 ppm, less than 400 ppm, less than 300 ppm, or even lessthan 250 ppm, 200 ppm, 150 ppm or 100 ppm.

The fermentation broth containing a salt of a long-chain dibasic acidrefers to a fermentation broth containing a salt of the long-chaindibasic acid produced during the biological fermentation for producingthe long-chain dibasic acid. The fermentation broth containing a salt ofa long-chain dibasic acid may contain sodium salt, potassium salt orammonium salt of the long-chain dibasic acid.

The sterilization is preferably membrane filtration: the residualbacteria and large proteins are separated by using a filtrationmembrane, and are effectively separated from the fermentation brothcontaining the salt of the long-chain dibasic acid. Further, the ceramicmembrane filtration process is preferred. When membrane filtration iscarried out using a ceramic membrane, it is preferred that thepre-membrane pressure is 0.2 to 0.4 MPa; preferably, the pore size ofthe filtration membrane is 0.05 to 0.2 μm.

The acidification is a treatment of acidifying the obtained membraneclear liquid containing a salt of a long-chain dibasic acid aftermembrane filtration, and the salt of the long-chain dibasic acid isconverted into a long-chain dibasic acid precipitate by adding an acid.It is preferred to use an inorganic acid such as sulfuric acid,hydrochloric acid, nitric acid, or mixture thereof for acidification.The inorganic acid during the acidification treatment is added in anamount sufficient to precipitate the long-chain dibasic acid in thesolution, mainly based on the endpoint pH of the solution, preferablythe acidification end point pH is lower than 5, and more preferablylower than 4.0. When an inorganic acid is added for acidification, thelong-chain dibasic acid precipitate and corresponding inorganic saltsolution can be obtained. The membrane clear liquid refers to the liquidobtained after membrane filtration by a membrane.

The solid-liquid separation is to separate the obtained long-chaindibasic acid precipitate from the acidified mother liquid, and thesolid-liquid separation includes filtration or/and centrifugation, and acommonly used solid-liquid separation device can be used.

Preferably, the step of extraction and purification further comprisesdecolorization of the fermentation broth containing a long-chain dibasicacid salt, adding activated carbon to the fermentation broth or themembrane clear liquid containing the salt of the long-chain dibasic acidfor decolorization treatment, and removing the activated carbon byfiltration after decolorization treatment. Decolorization step canfurther remove impurities in the long-chain dibasic acid solution.Preferably, the amount of activated carbon added is 0.1-5 wt %,preferably 1-3 wt % (relative to the amount of the long-chain dibasicacid contained in the solution).

The solvent crystallization, i.e., dissolving a long-chain dibasic acidprecipitate in an organic solvent, and crystallizing the long-chaindibasic acid by cooling, evaporation, and separating-out, and isolatingthe crystal to obtain a purified long-chain dibasic acid. The organicsolvent comprises one or more of alcohol, acid, ketone and ester;wherein the alcohol comprises one or more of methanol, ethanol,isopropanol, n-propanol and n-butanol; the acid comprises acetic acid;the ketone comprises acetone; and the ester comprises ethyl acetateand/or butyl acetate.

In another preferred embodiment, the long-chain dibasic acid precipitateis dissolved in an organic solvent, and then decolorized, and thenseparated to obtain a clear solution. When decolorized with activatedcarbon, the decolorization temperature is 85 to 100° C., and thedecolorization time is 15 to 165 min. In another preferred embodiment,after separating the clear liquid, cooling and crystallizing is carriedout, and cooling and crystallizing may include the steps of: firstcooling to 65-80° C., incubating for 1 to 2 hours, then cooling to25-35° C., and crystallizing. In another preferred embodiment, aftercrystallization, the resulting crystal is separated, thereby obtainingthe long-chain dibasic acid, and the crystal may be separated bycentrifugation.

In some embodiments, the present invention relates to the production ofnylon filaments, engineering plastics, synthetic fragrances,cold-resistant plasticizers, advanced lubricating oils, and polyamidehot melt adhesives using the dibasic acid products obtained above.

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur, and that thedescription comprises instances where the event or circumstance occursor does not occur. For example, “optionally a step” means that the stepis present or not present.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In some embodiments,the term “about” means within a standard deviation using measurementsgenerally acceptable in the art. In some embodiments, “about” means arange extending to +/−10% of the specified value.

The invention will be further illustrated by the following non-limitingexamples. Those skilled in the art will recognize that modifications canbe made to the invention without departing from the spirit thereof, andsuch modifications also fall within the scope of the invention.

The following experimental methods are all conventional methods unlessotherwise specified, and the experimental materials used can be easilyobtained from commercial companies unless otherwise specified.

Example 1 Culture Media, Culture Methods and Dibasic Acid DetectionMethod in the Examples

1. the formulation of YPD medium (w/v): 2% peptone, 2% glucose and 1%yeast extract (OXOID, LP0021). 1.5-2% agar powder was added to form asolid medium.

During culturing, a single colony was picked into a 2 mL centrifuge tubecontaining 1 mL YPD liquid medium, incubated at 30° C. on a shaker at250 RPM for 1 day.

2. the formulation of seed medium (w/v): sucrose 10-20 g/L (specificallyused, 10 g/L), yeast extract 3-8 g/L (specifically used, 3 g/L),industrial fermentation corn syrup (corn syrup for short, with totalnitrogen content of 2.5 wt %) 2-4 g/L (specifically used, 2 g/L), KH₂PO₄4-12 g/L (specifically used, 4 g/L), urea 0.5-4 g/L (specifically used,0.5 g/L) (separately sterilized at 115° C. for 20 min), and thefermentation substrate was n-dodecane, n-decane, and n-hexadecane, at 20mL/L, respectively.

During culturing, the cultured bacterial solution obtained in step 1 wasinoculated into a 500 mL shake flask containing 30 mL seed medium. Thebacterial solution was inoculated in an amount of 3-5% and incubated at30° C. on a shaker at 250 RPM until OD₆₂₀ reached 0.8 (30-folddilution).

3. Fermentation medium (w/v): sucrose 10-40 g/L (specifically used, 10g/L), corn syrup (total nitrogen content of 2.5 wt %) 1-5 g/L(specifically used, 1 g/L), yeast extract 4-12 g/L (specifically used, 4g/L), NaCl 0-3 g/L (not used), KNO₃ 4-12 g/L (specifically used, 4 g/L),KH₂PO₄ 4-12 g/L (specifically used, 4 g/L), urea 0.5-3 g/L (specificallyused, 0.5 g/L) (separately sterilized at 115° C. for 20 min), and thefermentation substrate was n-dodecane, n-decane, and n-hexadecane, at300-400 mL/L (specifically used, 300 mL/L), respectively, and acrylicacid 4 g/L, and 1N HCl and 1N NaOH was used to adjust pH to 7.5-7.6.

In fermentation, the seed solution obtained in step 2 was inoculatedinto a 500 mL shake flask containing 15 mL fermentation medium. Theamount of inoculum is 10-30% and incubated at 30° C. on a shaker at 250RPM for 90-144h. During culturing, the pH value was adjusted to thespecified range by adding acid or base at a certain interval of time.

4. Steps for determining the yield of dibasic acid and the content ofmonobasic acid impurity by gas chromatography (GC)

(1) Detection of fermentation broth product and impurity content: Thefermentation broth was pretreated by conventional gas chromatography anddetected by gas chromatography. The chromatographic conditions were asfollows:

Column: Supelco SPB-50 30m*0.53 mm*0.5 μm (Cat. No. 54983).

Gas Chromatograph (Shimadzu, GC-2014).

Method: The initial temperature was 100° C., and the temperature wasraised to 230° C. at a rate of 15° C./min and kept for 2 min. Thecarrier gas was hydrogen, the inlet temperature was 280° C., the FIDtemperature was 280° C., and the injection volume was 4 μL.

The yield of the dibasic acid was calculated based on the ratio of thepeak area of the dibasic acid product and the internal standard peakarea with a known concentration, and the impurity content was calculatedby the ratio of the peak area of the dibasic acid product to the peakarea of the impurity.

(2) Determination of purity and impurity content of solid product: thesolid product was pretreated by conventional gas chromatography anddetected by gas chromatography,

Chromatographic conditions: Column: Supelco SPB-50 30 m*0.53 mm*0.5 μm(Cat. No. 54583).

Gas Chromatograph (Shimadzu, GC-2014).

Method: The initial temperature was 100° C., and the temperature wasraised to 230° C. at a rate of 15° C./min and kept for 2 min. Thecarrier gas was hydrogen, the inlet temperature was 280° C., the FIDtemperature was 280° C., and the injection volume was 4 μL.

The purity of the product and impurity content were calculated from thepeak area of the dibasic acid product and the peak area of impurity.

Example 2 Preparation of CYP52A12 Mutation Template

1. Preparation of CYP52A12 Promoter Mutation Template

The genomic DNA of Candida tropicalis CCTCC M2011192 was extracted byusing Ezup Yeast Genomic DNA Extraction Kit (Sangon, Cat No. 518257).The method with liquid nitrogen grinding was used in favor of increasingthe cell wall disruption efficiency. Genomic DNA obtained by this methodwas used as template for error-prone PCR. The obtained mutation-freeproduct was called CYP52A12 and was confirmed by sequencing to beidentical to the sequence set forth by GenBank Accession Number:AY230498.

2. Error-Prone PCR

The concentration of Mg²⁺ was adjusted (2-8 mM, increasing by 0.5 mM)and the promoter of CYP52A12 gene was amplified by error-prone PCR usingnormal Taq enzyme (Takara, Cat No. R001B). The primers were as follows:

(SEQ ID NO. 1) Pcyp52a12-F: 5′- GTCGACTTCTCCTTTAGGCA-3′ (SEQ ID NO. 2)Pcyp52a12-R: 5′- TCGATGATTTCTTGTGTGGC-3′

PCR reaction conditions were:

Step 1: 98° C. for 30 s,

Step 2: 98° C. for 10 s, 52° C. for 30 s, 72° C. for 1 m, 35 cycles,

Step 3: 72° C. for 5 m.

The PCR product was subjected to 1% agarose gel electrophoresis andrecovered and purified by using the Axygen Gel Recovery Kit (Axygen,AP-GX-250G).

Example 3 Preparation of Homologous Recombination Template

All DNA fragments in this example were obtained by amplification usingPrimeSTAR® HS High Fidelity DNA polymerase (Takara, R040A). The DNAfragments were subjected to 1% agarose gel electrophoresis, followed byrecovery and purification by using the Axygen Gel Recovery Kit.

(1) Amplification of the upstream and downstream homologousrecombination fragments. The template was the above genomic DNA ofCandida tropicalis. The primer sequences were as follows:

(SEQ ID NO. 3) CYP52A12_Upstream-F: 5′- GGTCGAGGAAGTGGCATTAAA-3′(SEQ ID NO. 4) CYP52A12_Upstream-R: 5′- ACCTCCTGCAGTTGCCAT-3′

The PCR reaction conditions were as follows:

Step 1: 98° C. for 30 s,

Step 2: 98° C. for 10 s, 53° C. for 10 s, 72° C. for 30 s, 30 cycles,

Step 3: 72° C. for 5 m.

The resultant product was designated as CYP52A12_Upstream, and verifiedby sequencing, set forth in SEQ ID NO. 17.

(SEQ ID NO. 5) CYP52A12_Downstream-F: 5′- ATGGCCACACAAGAAATCAT-3′(SEQ ID NO. 6) CYP52A12_Downstream-R: 5′- AGTCTGGGAGTAACTTCTGG-3′ .

The PCR reaction conditions were as follows:

Step 1: 98° C. for 30 s,

Step 2: 98° C. for 10 s, 50° C. for 10 s, 72° C. for 45 s, 30 cycles,

Step 3: 72° C. for 5 m.

The resultant product was designated as CYP52A12_Downstream, andverified by sequencing, the sequence of which was set forth in SEQ IDNO. 18.

(2) Amplification of the resistance screening marker (HYG, hygromycinresistance gene). The amplification template was the vector pCIB2 (SEQID NO. 11) owned by our company. The primer sequences were as follows:

CYP52A12 HYG-F: (SEQ ID NO. 7)5′-ATGGCAACTGCAGGAGGTGCATGCGAACCCGAAAATGG-3′ CYP52A12 HYG-R:(SEQ ID NO. 8) 5′-TGCCTAAAGGAGAAGTCGACGCTAGCAGCTGGATTTCACT-3′ .

The PCR reaction conditions were as follows:

Step 1: 98° C. for 30 s,

Step 2: 98° C. for 10 s, 55° C. for 10 s, 72° C. for 1 m 50 s, 5 cycles,

Step 3: 98° C. for 10 s, 72° C. for 2 m, 25 cycles,

Step 4: 72° C. for 5 m.

The resultant product, called HYG, was verified by sequencing, as setforth in SEQ ID NO. 9.

(3) PCR overlap extension to obtain a complete recombination template.

The four PCR fragments recovered above were subjected to overlapextension to obtain a homologous recombination template, which wasrecovered and purified. The specific method was as follows:

Overlap extension PCR was performed by adding an equimolar amount of thefragments CYP52A12_Upstream, Pcyp52a12, HYG and CYP52A12_Downstream astemplates, using primers CYP52A12_Upstream-F and CYP52A12_Downstream-R,and using PrimeSTAR® HS High Fidelity DNA polymerase.

The PCR reaction conditions were as follows:

Step 1: 98° C. for 30 s,

Step 2: 98° C. for 10 s, 55° C. for 10 s, 72° C. for 5 m 30 s, 30cycles,

Step 3: 72° C. for 8 m.

The recombination fragment with a size of approximately 5.1 Kb wasrecovered and purified after gel electrophoresis.

FIG. 1 is a schematic diagram of the integration of the CYP52A12 genewith a mutation site by the homologous recombination and removal of thehygromycin resistance marker according to the present invention.

Example 4 Construction of Candida tropicalis CYP52A12 Gene MutantLibrary

1. Preparation of Yeast Electroporation-competent Cells

The yeast cells CCTCC M2011192 subjected to overnight incubation at 30°C. on a shaker at 250 RPM were inoculated into 100 mL of the YPD mediumof Example 1 to OD₆₂₀ of 0.1, and cultured under the same conditions toOD₆₂₀ of 1.3. The cells were collected by centrifugation at 3000 g, 4°C. Cells were washed twice with ice-cold sterile water and collected,and then the cells were re-suspended in 10 mL of ice-cold 1M sorbitolsolution. The cells were collected by centrifugation at 4° C., 1500 gand re-suspended in 1 mL sorbitol solution above. Aliquots of 1004 ofcell suspension were for genetic transformation.

2. Competent Yeast Cell Electroporation

1 μg of the DNA fragments for recombination recovered in step (3) ofExample 3 were added to the above competent cells, and placed on ice for5 min and transferred to a 0.2 cm cuvette, and then performingelectroporation (BioRad, Micropulser™ Electroporator, program SC2, 1.5kV, 25 uFD, 200 ohms). A mixture of 1 mL of YPD and 1M sorbitol (1:1,v/v) was quickly added, and cultured at 30° C., 200 RPM for 2 hours. Thebacterial cells were collected and plated on a YPD medium plate with 100mg/L of hygromycin B, placed still at 30° C. for 2-3 days until singlecolonies appeared.

Example 5 Screening of Mutant Strains

1. Screening method: single colonies obtained in Example 4 were pickedinto a 2 mL centrifuge tube with 1 mL YPD medium of Example 1(containing 100 mg/L hygromycin B), and cultured at 30° C. on a shakerat 250 RPM for 1 day. The above bacterial solution was inoculated into a500-mL shake flask with 30 mL of the seed medium of Example 1(containing 100 mg/L hygromycin B). The inoculum amount was 3%, culturedat 250 RPM and 30° C. until OD₆₂₀ reached 0.8 (30-fold dilution). Theseed solution was inoculated into a 500-mL shake flask containing 15 mLof the fermentation medium of Example 1, the inoculum amount was 20%,and the substrate was n-dodecane in the fermentation medium. The cultureat 250 RPM and 30° C. was continued until the end of the fermentation.The original strain CCTCC M2011192 was used as control: the medium,culture and fermentation methods were the same as above except that themedium did not contain hygromycin B.

0.5 g sample of the above fermentation broth was taken and subjected toGC assay using the method described in Example 1 (4), and the content ofC12 dibasic acid content and the mass ratio of the monobasic acidimpurity having 12 carbon atoms were calculated.

2. Screening results: a candidate strain with a significant reduction inmonobasic acid impurity content compared to the original strain CCTCCM2011192 was screened, designated as 430HYG, and the results were shownin Table 1 below.

TABLE 1 Control CCTCC Strain M2011192 430 HYG Yield of C12 dibasic acid(mg/g) 148.8 149.6 Mass ratio of monobasic acid impurity 2.22 1.15having 12 carbon atoms (%)

The mass ratio of the monobasic acid impurity of the present inventionwas the mass percentage of it to C12 dibasic acid. From Table 1, themass ratio of the monobasic acid impurity having 12 carbon atoms wasdecreased by 48.2%.

Example 6 Sequence Analysis of CYP52A12 Gene in the Mutant Strain

1. According to the method of Example 2, the genomic DNAs of the yeastCCTCC M2011192 and 430HYG were extracted, and the promoter region of theCYP52A12 gene was amplified using PrimeSTAR® HS High Fidelity DNApolymerase (Takara). The primers were CYP52A12-F and CYP52A12-R.

The PCR reaction conditions were as follows:

Step 1: 98° C. for 30 s,

Step 2: 98° C. for 10 s, 52° C. for 10 s, 72° C. for 1 m, 30 cycles,

Step 3: 72° C. for 5 m.

2. After completion of the PCR, the product was subject to gelelectrophoresis and recovered and purified.

3. Addition of As to the purified PCR fragment: 204 of recovered PCRamplified fragment was added to 4 μL of 10× Takara Taq Buffer, 3.2 μL ofdNTP (each 10 mM) and 0.2 μL of Takara Taq, supplemented with ddH₂O to40 μL, incubated at 72° C. for 20 minutes, and recovered by Axygen PCRpurification kit.

4. TA cloning. 4 μL of the PCR fragment recovered after addition of Aswere added to 1 μL pMD19-T vector backbone and 5 μL Solution I, mixedwell and incubated at 16° C. for 30 min. The ligation product wastransformed into DH5a chemical competent cells and positive clones werepicked and sent to Majorbio for sequencing.

The results showed that: the sequence of the CYP52A12 gene of theparental CCTCC M2011192 was identical to the sequence in the GenBANKdatabase (Accession Number: AY230498), while the mutant strain 430HYGhad base mutations in the promoter region. As shown in FIG. 2, severalbase mutations occurred in the promoter region of CYP52A12 (indicatedwith black box in the sequence alignment result), the sequence of whichwas set forth in SEQ ID NO: 16.

Example 7 Removal of the Resistance Marker

1. Preparation of Homologous Recombination Template

The genomic DNA of the Candida tropicalis mutant strain 430HYG was usedas a template to amplify recombinant template fragmentsCYP52A-Upstream-2 and Pcyp52a12 necessary for removal of the resistancescreening marker, using PrimeSTAR® HS high-fidelity DNA polymerase, andrecovered after gel electrophoresis. The sequence obtained was set forthin SEQ ID NOs. 14 and 15. The primer sequences and PCR reactionconditions were as follows:

CYP52A12_Upstream-F: (SEQ ID NO. 3) 5′-GGTCGAGGAAGTGGCATTAAA-3′CYP52A12_Upstream-2R: (SEQ ID NO. 10)TGCCTAAAGGAGAAGTCGACACCTCCTGCAGTTGCCAT-3′ Pcyp52a12-F: (SEQ ID NO. 1)5′-GTCGACTTCTCCTTTAGGCA-3′ Pcyp52a12-R: (SEQ ID NO. 2)5′-TCGATGATTTCTTGTGTGGC-3′

The PCR reaction conditions were as follows:

Step 1: 98° C. for 30 s,

Step 2: 98° C. for 10 s, 52° C. for 10 s, 72° C. for 1 m, 30 cycles,

Step 3: 72° C. for 5 m.

The above PCR fragments were recovered and purified, and equimolaramounts of CYP52A12_Upstream-2 and Pcyp52a12 were added as templates,with the primers of CYP52A12_Upstream-F and Pcyp52a12-R. PCR overlapreaction was carried out with PrimeSTAR® HS high-fidelity DNApolymerase. The PCR conditions were as follows:

Step 1: 98° C. 30 s

Step 2: 98° C. 10 s, 52° C. 10 s, 72° C. 1 m 30 s; 30 cycles

Step 3: 72° C. 5 m.

After gel electrophoresis, a fragment of about 1.3 Kb obtained by theoverlap extension was recovered and purified, i.e. a homologousrecombination template necessary for the removal of the hygromycinscreening marker, the sequence of which was set forth in SEQ ID NO. 12.

2. Removal of the Resistance Marker

Freshly electro-competent cells of the strain 430HYG were prepared and 1μg of the recombination fragment recovered in step 1 was added. Afterbeing placed on ice for 5 min, the cells were quickly transferred to apre-chilled 0.2 cm cuvette on ice and transformed by electroporation(supra, 1.5 kV, 25 uFD, 200 ohms). A mixture of 1 mL YPD and 1M sorbitol(1:1, v/v) was quickly added, and incubated at 30° C. and 200 RPM for 2hours. The bacterial cells were collected and plated on an YPD mediumplate without an antibiotic, and cultured at 30° C. for 2-3 days untilsingle colonies appeared.

3. Screening for Strains with the Resistance Marker Removed

Single colonies were picked and correspondingly inoculated on YPD plateswith and without hygromycin (100 mg/L). Single colonies that could growon the medium without the antibiotic but could not grow on the mediumwith the antibiotic were picked and inoculated to a 2 mL centrifuge tubecontaining 1 mL of the YPD medium, incubated overnight at 4° C. and 250RPM, and the colony PCR was used to determine whether the resistancescreening marker was removed or not in the next day. The DNA polymeraseused was Takara Taq, with the primers:

a) CYP52A12_Upstream-F and Pcyp52a12-R,

b) HYG-F: (SEQ ID NO. 19) 5′-CTCGGAGGGCGAAGAATCTC-3′ HYG-R:(SEQ ID NO. 20) 5′-CAATGACCGCTGTTATGCGG-3′.

The PCR conditions were as follows:

Step 1: 98° C. for 30 s,

Step 2: 98° C. for 10 s, 52° C. for 30 s, 72° C. for 35 s, 30 cycles,

Step 3: 72° C. for 5 min.

4. Screening Results

By colony PCR, one strain with the resistance screening marker removedwas screened out, and verified by sequencing that the same mutationswere present in the promoter region of the CYP52A12 gene in this strainas the strain 430HYG, and the hygromycin screening marker gene wasremoved. This strain was eventually designated as 430.

Example 8 Fermentation Production of Long-Chain Dodecanedioic Acid byStrain 430

Fermentation: Strain 430 was inoculated to a 2 mL centrifuge tubecontaining 1 mL of YPD medium of Example 1, and incubated at 30° C. on ashaker at 250 RPM for 1 day. The above bacterial solution was inoculatedinto a 500-mL shake flask with 30 mL of the seed medium of Example 1,wherein the inoculation amount was 3%, and cultured at 250 RPM and 30°C. on a shaker until OD₆₂₀ reached 0.8 (after 30-fold dilution). Theseed solution was inoculated into a shake flask containing 15 mL of thefermentation medium of Example 1, wherein the inoculation amount was20%, and the substrate was n-dodecane in the fermentation medium. Theculturing on shaker at 250 RPM and 30° C. was continued until thecompletion of the fermentation. The strain CCTCC M2011192 was used ascontrol, and the medium, culture and fermentation methods were the sameas described above.

A 0.5 g sample of the fermentation broth was taken and measured by GCdetection using the method described in Example 1 (4), and the yield ofC12 dibasic acid and the mass ratio of the monobasic acid impurityhaving 12 carbon atoms were calculated. The results were shown in Table2 below.

TABLE 2 Strain CCTCC M2011192 430 Yield of C12 dibasic acid (mg/g) 150.4151.3 Mass ratio of the monobasic 2.16 1.09 acid impurity having 12carbon atoms (%)

It can be seen from Table 2 that the mass ratio of the monobasic acidimpurity having 12 carbon atoms was decreased by 49.5% after the removalof the resistance marker.

Extraction and Purification:

(1) The pH of the above fermentation broth was adjusted to 8.5 with 30%(mass concentration) sodium hydroxide solution, the concentration oflong-chain dibasic acid was adjusted by adding water to 8.9 wt % andheated to 45° C., and the fermentation broth was filtered with a ceramicmembrane with pore size of 0.05 μm (purchased from Suntar MembraneTechnology (Xiamen) Co., Ltd.). The area of the ceramic membrane usedwas 0.84 square meters, and the pre-membrane pressure was set to 0.3MPa. The membrane clear liquid was collected.

(2) The obtained membrane clear liquid was decolorized by adding 5 wt %of powdered activated carbon (relative to the amount of long-chaindibasic acid contained in the solution) at 60° C., and filtered toobtain a clear liquid.

(3) The clear liquid was further added with sulfuric acid, the pH wasadjusted to 3.5, cooled to 30° C., and filtered to obtain a wet solid.The filter cake was washed with pure water the weight of which was 3times to the wet solid, and filtered and dried to obtain primary C12dibasic acid product.

(4) Acetic acid at a concentration of 97% whose amount was 3.5 timesrelative to the weight of the primary C12 dibasic acid product was addedto the primary C12 dibasic acid product and heated to 85° C. todissolve, and 1% macroporous powdered activated carbon (relative to theweight of the primary C12 dibasic acid product) was added fordecolorization and kept at 85° C. for 1 hour, and hot-filtered to obtaina clear liquid. The temperature of the solution was reduced at a rate of10° C./hour to obtain a long-chain dibasic acid crystal solution at 30°C. The solution was filtered and the solvent of the wet solid was washedwith water, and dried to obtain secondary C12 dibasic acid product.

(5) Step (4) was repeated on the secondary C12 dibasic acid product toobtain the tertiary C12 dibasic acid product.

The purity of the C12 dibasic acid product and the content of monobasicacid impurity obtained in extraction and purification steps (3)-(5) weredetermined and calculated using the method described in Example 1 (4),as shown in Table 3 below:

TABLE 3 CCTCC Dodecanedioic acid Strain M2011192 430 Primary productPurity of C12 dibasic 97.32 98.21 acid (%) Content of monobasic 112006400 acid impurity having 12 carbon atoms (ppm) Secondary product Purityof C12 dibasic 98.55 99.02 acid (%) Content of monobasic 740 475 acidimpurity having 12 carbon atoms (ppm) Tertiary product Purity of C12dibasic 99.72 99.85 acid (%) Content of monobasic 365 165 acid impurityhaving 12 carbon atoms (ppm)

Example 9 To further verify the above mutations, the genomic DNA ofyeast 430HYG was extracted, and the DNA fragment containing the mutatedCYP52A12 and HYG resistance gene was amplified via PCR using PrimeSTAR®HS high-fidelity DNA polymerase, with the primers CYP52A12_Upstream-F(SEQ ID NO. 3) and Pcyp52a12-R (SEQ ID NO. 2), and the PCR reactionconditions were as follows:

Step 1: 98° C. 30 s

Step 2: 98° C. 10 s, 52° C. 10 s, 72° C. 4 m, 30 cycles

Step 3: 72° C. 5 m.

The fragment with a size of approximately 4 Kb was recovered andpurified after gel electrophoresis, and verified by sequencing, thesequence of which was set forth in SEQ ID NO. 13. The process ofintroducing via homologous recombination the above DNA fragment into thestrain CCTCC M2011192 was the same as in Examples 4 and 5, and thesequencing procedure of the promoter of the CYP52A12 gene of the singleclone obtained by screening was the same as in Example 6. It wasverified by sequencing that the selected single clone was integratedwith the CYP52A12 gene with mutations, and the mutation sites wereconsistent with SEQ ID NO. 16. One of the bacterial strains was named as431HYG.

The fermentation method was the same as described in Example 5, and thestrains used were CCTCC M2011192, 430HYG and 431HYG. After thefermentation, the samples of 0.5 g of the above fermentation broths weretaken to calculate the yield of C12 dibasic acid and the content ofmonobasic acid impurity, as shown in Table 4. The results showed that,consistent with 430HYG, the content of monobasic acid impurity in 431HYGwas significantly reduced compared to that of the control CCTCCM2011192.

TABLE 4 CCTCC Strain M2011192 430HYG 431HYG Yield of C12 dibasic acid(mg/g) 149.8 150.2 150.1 Mass ratio of monobasic acid 2.17 1.10 1.11impurity having 12 carbon atoms (%)

Example 10 Determination of the Influence of Different Base MutationSites on the Content of Monobasic Acid Impurity

1. Synthesis of the Promoter Sequences Containing Different Combinationsof Mutation Sites

A promoter sequence containing different combinations of mutation sites(Genbank Accession No. AY230498) was synthesized using the whole-genomesynthesis (Sangon) conventionally used in the art, comprising 912 bpupstream of the start codon and adjacent 23 bp CDS sequence. Taking thefirst base upstream of the start codon as −1, the specific mutationsites were as follows:

Pcyp52a12-1: −7_−4delACCA;

Pcyp52a12-2: −412_−411AC>TT, −402insTT, −7_−4delACCA;

Pcyp52a12-3: −579A>G, −412_−411AC>TT, −402insTT, −7_−4delACCA;

Pcyp52a12-4: −831delT, −825C>A, −823delG, −579A>G, −412_−411AC>TT,−402insTT, −7_−4delACCA (corresponding to −15_1ACCAACCAACCAACCA (SEQ IDNO.: 37)>ACCAACCAACCA). (SEQ ID NO.: 38)

2. Preparation of Homologous Recombination Template

The homologous recombination template was prepared in the same manner asin Example 3 except that the promoter fragment was different. After PCR,the DNA fragment with a size of about 5.1 Kb, i.e. homologousrecombination template, was recovered and purified, and verified bysequencing, designated as T12-1, T12-2, T12-3 and T12-4 in the orderdescribed in step 1 of Example 10, successively.

3. Construction of Mutant Strains Containing Different Combinations ofMutation Sites in the Promoter Region of CYP52A12

The mutant strains containing different combinations of mutation sitesin the CYP52A12 promoter region were constructed by homologousrecombination method. The method was the same as in Examples 4 and 5.After 2-3 days, the colonies growing on the plate were picked andidentified by colony PCR. The results of the recombination were verifiedby sequencing. The method was the same as in Example 6. Afterverification by sequencing, one strain of the obtained mutant strainswas randomly selected and designated as P12-1, P12-2, P12-3 and P12-4 inthe order described in step 1 of Example 10, respectively.

4. Comparison of Long Chain C12 Dibasic Acids Produced by Fermentationof Different Strains

Single colonies of 430HYG, P12-1, P12-2, P12-3 and P12-4 were picked andinoculated into a 2 mL centrifuge tube with 1 mL of the YPD medium(containing 100 mg/L hygromycin B) in Example 1, incubated in a shakerat 250 RPM and 30° C. for 1 day. The above bacterial solution was takeninto a 500 mL shake flask containing 30 mL of the seed medium of Example1, wherein the inoculation amount was 3%, and incubated at 250 RPM and30° C. until the OD₆₂₀ reached 0.8 (after 30-fold dilution). The seedsolution was inoculated into a 500 mL shake flask containing 15 mL ofthe fermentation medium described in Example 1, in an amount of 20%, andthe substrate in the fermentation medium was n-dodecane, and continuedto culture at 250 RPM, 30° C. until the end of the fermentation. Theoriginal strain CCTCC M2011192 was used as control: the medium, cultureand fermentation methods were the same as above, except that the YPDmedium did not contain hygromycin B.

After the fermentation, the samples of 0.5 g of the fermentation brothswere subjected to GC detection according to the method described in Step4 of Example 1, and the yield of C12 dibasic acid and the mass ratio ofmonobasic acid impurity having 12 carbon atoms to C12 dibasic acid werecalculated. The results were shown in Table 5 below.

TABLE 5 Mass ratio of monobasic Yield of C12 acid having 12 carbondibasic acid Strain atoms (%) (mg/g) CCTCC231192 2.20 148.7 430HYG 1.14149.5 P12-1 1.96 148.5 P12-2 1.32 149.1 P12-3 1.20 148.9 P12-4 1.21149.6

From the data of the content of monobasic acid impurity in Table 5, itcan be inferred that the mutation site which significantly reduced thecontent of the monobasic acid impurity having 12 carbon atoms in thefermentation broth includes −579A>G, −412_−411AC>TT, −402insTT and−7_−4delACCA. Changes in these sites may affect the binding of atranscription factor to the cis-acting element of the promoter region,thereby affecting the transcription of the CYP52A gene.

Example 11 Fermentation of Strain 430 to Produce Long-Chain C10 DibasicAcid

Fermentation: Strain 430 was inoculated to a 2 mL centrifuge tubecontaining 1 mL of YPD medium of Example 1, incubated at 30° C. on ashaker at 250 RPM for 1 day. The above bacterial solution was inoculatedinto a 500-mL shake flask with 30 mL of the seed medium of Example 1,wherein the inoculation amount was 3%, and cultured at 250 RPM and 30°C. for 36-48h until OD₆₂₀ reached 0.8 (after 30-fold dilution). The seedsolution was inoculated into a shake flask containing 15 mL of thefermentation medium of Example 1, wherein the inoculation amount was20%, and the substrate was n-decane in the fermentation medium. Theculture on a shaker at 250 RPM and 30° C. was continued until the end ofthe fermentation. The strain CCTCC M2011192 was used as control, and themedium, culture and fermentation methods were the same as describedabove.

After fermentation, 0.5 g sample of the fermentation broth was taken andsubjected to GC detection according to the method as described inExample 1 (4). The yield of C10 dibasic acid and the content ofmonobasic acid impurity of mutant strain were compared with the parentalstrain, and the results were shown in Table 6 below.

TABLE 6 Strain CCTCC M2011192 430 Yield of C10 dibasic acid (mg/g) 122.5125.6 Mass ratio of monobasic acid 1.55 0.79 impurity having 10 carbonatoms (%)

It can be seen from Table 6 that the mass ratio of monobasic acidimpurity having 10 carbon atoms was decreased by 49.0%.

Extraction and purification steps: they were the same as the extractionand purification steps of Example 8, except for the absence of step (5).The purity of the primary and secondary C10 dibasic acid product and thecontent of monobasic acid impurity were determined and calculated usingthe method described in Example 1 (4), as shown in Table 7 below:

TABLE 7 C10 dibasic acid Strain CCTCC M2011192 430 Primary productPurity of C10 dibasic 98.18 99.07 acid (%) Content of monobasic 37501835 acid impurity having 10 carbon atoms (ppm) Secondary Purity of C10dibasic 98.90 99.85 product acid (%) Content of monobasic 535 280 acidimpurity having 10 carbon atoms (ppm)

Example 12 Production of Long-Chain C16 Dibasic Acid by Fermentation ofthe Strain 430

Fermentation: Strain 430 was inoculated to a 2 mL centrifuge tubecontaining 1 mL of YPD medium of Example 1, and incubated at 30° C. on ashaker at 250 RPM for 1 day. The above bacterial solution was inoculatedinto a 500-mL shake flask with 30 mL of the seed medium of Example 1,wherein the inoculation amount was 3%, and cultured at 250 RPM and 30°C. for 36-48h until OD₆₂₀ reached 0.8 (after 30-fold dilution). The seedsolution was inoculated into a shake flask containing 15 mL of thefermentation medium of Example 1, wherein the inoculation amount was20%, and the substrate was n-hexadecane in the fermentation medium. Theculture on a shaker at 250 RPM and 30° C. was continued until the end ofthe fermentation. The strain CCTCC M2011192 was used as control, and themedium, culture and fermentation methods were the same as describedabove.

After the fermentation, 0.5 g sample of the fermentation broth was takenand subjected to GC detection according to the method described inExample 1 (4). The yield of C16 dibasic acid and the content ofmonobasic acid impurity of mutant strain were compared with the parentalstrain, and the results were shown in Table 8 below.

TABLE 8 Strain CCTCC M2011192 430 Yield of C16 dibasic acid (mg/g) 125.1126.6 Mass ratio of monobasic acid 5.06 2.89 impurity having 16 carbonatoms (%)

It can be seen from Table 8 that the mass ratio of monobasic acidimpurity having 16 carbon atoms was decreased by 42.9%.

Extraction and purification steps were the same as the extraction andpurification steps of Example 8, except for the absence of step (5). Thepurity of the primary and secondary C16 dibasic acid product as well asthe content of monobasic acid impurity were determined and calculatedusing the method described in Example 1 (4), as shown in Table 9 below:

TABLE 9 C16 dibasic CCTCC acid Strain M2011192 430 Primary Purity of C16dibasic acid (%) 81.10 83.50 product Content of monobasic acid impurity13650 10240 having 16 carbon atoms (ppm) Secondary Purity of C16 dibasicacid (%) 98.40 99.15 product Content of monobasic acid impurity 62053470 having 16 carbon atoms (ppm)

Example 13

The DNA fragment (SEQ ID NO: 13) in Example 9 was introduced intoCandida tropicalis (CCTCC M 203052) by homologous recombinationaccording to the methods as described in Examples 4 and 5. The methodfor sequencing the promoter of the gene CYP52A12 of the obtained singlecolony and the parent strain (CCTCC M 203052) were the same as describedin Example 6. By sequencing, it was confirmed that the sequence of thegene CYP52A12 in the parent strain (CCTCC M 203052) was consistent withthe published sequence in GenBank with the Accession Number of AY230498,while the screened out colony carried a mutation in this gene in whichthe mutation was consistent with SEQ ID NO: 16. One strain wasdesignated as 432HYG.

The method for fermentation was according to Example 5, in which thestrains used were CCTCC M 203052 and 432HYG. After completion offermentation, a sample of 0.5 g of each fermentation broth wascollected, and the yield of C12 dibasic acid and the amount of monobasicacid impurity having 12 carbon atoms were calculated, as shown in Table10. The results indicated that the content of the monobasic acidimpurity in the fermentation broth by the strain 432HYG wassignificantly reduced compared with the parent strain CCTCC M 203052.

TABLE 10 Strain CCTCC M203052 432HYG Yield of C12 dibasic acid 135.8134.1 (mg/g) Mass ratio of monobasic 1.23 0.59 acid impurity having 12carbon atoms (%)

From the above Examples 8-13 regarding the fermentation production oflong-chain dibasic acids from different fermentation substrates, it canbe seen that the content of monobasic acid impurity in the fermentationbroth after fermentation was significantly reduced; compared to theparental strain, the content of monobasic acid impurity could be reducedby more than 40%, and sometimes reduced by nearly 50%, and furtherextraction and purification of the obtained C12, C10 and C16 dibasicacids could further reduce the content of monobasic acid impurity, whichreduces the difficulty of the later extraction and purificationprocesses to a great extent.

The invention claimed is:
 1. A product, which is one of the products I)and II): I) an isolated DNA molecule, which, corresponding tonucleotides 1-1176 or 265-1176 of SEQ ID NO: 21, wherein SEQ ID NO: 21comprises a gene encoding a CYP52A12 protein, comprises any one or moreof base mutations 301A>G, 324A>T, 346delT, 352C>A, 354delG, 598A>G,765_766AC>TT, 774insTT and 1162_1176ACCAACCAACCAACC (SEQ ID NO:39)>ACCAACCAACC (SEQ ID NO: 40), wherein the promoter region, theisolated DNA molecule has at least 95% sequence identity to SEQ ID NO:16; and II) a microorganism containing the isolated DNA molecule of I),which produces a long-chain dibasic acid with decreased content ofmonobasic acid impurity, compared to a microorganism not containing theisolated DNA molecule of I), wherein the decreased content of monobasicacid impurity is more than 0, and less than 12,000 parts per million(ppm), 10,000 ppm, 6,000 ppm, 3,000 ppm, 1,000 ppm, 500 ppm, or 200 ppmor less.
 2. The product of claim 1, which is I) the isolated DNAmolecule, wherein the isolated DNA molecule comprises: (ii) basemutations 765_766AC>TT, 774insTT and 1162_1176ACCAACCAACCAACC (SEQ IDNO: 39)>ACCAACCAACC (SEQ ID NO: 40); or (vi) a sequence comprising thenucleotide sequence set forth in any of SEQ ID NOs: 27-36.
 3. Theproduct of claim 1, which is II) the microorganism, wherein themicroorganism is: (i) selected from the group consisting ofCorynebacterium, Geotrichum candidum, Candida, Pichia, Rhodotroula,Saccharomyces and Yarrowia; (ii) yeast; or (iii) Candida tropicalis orCandida sake.
 4. The product of claim 1, which is II) the microorganism,wherein the long-chain dibasic acid is: (i) selected from the groupconsisting of C9 to C22 long-chain dibasic acids; (ii) selected from thegroup consisting of C9 to C18 long-chain dibasic acids; (iii) one ormore selected from the group consisting of C10 dibasic acid, C11 dibasicacid, C12 dibasic acid, C13 dibasic acid, C14 dibasic acid, C15 dibasicacid and C16 dibasic acid; (iv) at least one or more of C10 to C16dibasic acids, or at least one or more of n-C10 to C16 dibasic acids; or(v) at least one or more selected from the group consisting of sebacicacid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid,tetradecanedioic acid, pentadecanedioic acid and hexadecanedioic acid.5. The product of claim 1, which is II) the microorganism, wherein themonobasic acid impurity: (i) comprises those having the chemical formulaof CH₃—(CH₂)n-COOH, where n≥7, and/or CH₂OH—(CH₂)n-COOH, where n≥7; (ii)comprises, but not limited to, a long-chain monobasic acid with thenumber of carbon atoms in the carbon chain greater than 9; or (iii)comprises any one or more selected from the group consisting of amonobasic acid having 9 carbon atoms, a monobasic acid having 10 carbonatoms, a monobasic acid having 11 carbon atoms, a monobasic acid having12 carbon atoms, a monobasic acid having 13 carbon atoms, a monobasicacid having 14 carbon atoms, a monobasic acid having 15 carbon atoms, amonobasic acid having 16 carbon atoms, a monobasic acid having 17 carbonatoms, a monobasic acid having 18 carbon atoms, and a monobasic acidhaving 19 carbon atoms.
 6. A method of producing a long-chain dibasicacid or fermentation broth comprising long chain dibasic acid,comprising culturing the microorganism of claim 1 II), a microorganismcontaining the isolated DNA molecule of claim 1 I), which produces along-chain dibasic acid with decreased content of monobasic acidimpurity, compared to a microorganism not containing the isolated DNAmolecule of claim 1 I), and in the method of producing a long chaindibasic acid, isolating, extracting and/or purifying the long-chaindibasic acid from the culture.
 7. The method of claim 6, wherein themicroorganism is: (i) selected from the group consisting ofCorynebacterium, Geotrichum candidum, Candida, Pichia, Rhodotroula,Saccharomyces and Yarrowia; (ii) yeast; or (iii) Candida tropicalis orCandida sake.
 8. The method of claim 6, wherein the long-chain dibasicacid is: (i) selected from the group consisting of C9 to C22 long-chaindibasic acids; (ii) selected from the group consisting of C9 to C18long-chain dibasic acids; (iii) one or more selected from the groupconsisting of C10 dibasic acid, C11 dibasic acid, C12 dibasic acid, C13dibasic acid, C14 dibasic acid, C15 dibasic acid and C16 dibasic acid;(iv) at least one or more of C10 to C16 dibasic acids, or at least oneor more of n-C10 to C16 dibasic acids; or (v) at least one or moreselected from the group consisting of sebacic acid, undecanedioic acid,dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid,pentadecanedioic acid and hexadecanedioic acid.
 9. The method of claim6, wherein the monobasic acid impurity: (i) comprises those having achemical formula of CH₃—(CH₂)n-COOH, where n≥7, and/orCH₂OH—(CH₂)n-COOH, where n≥7; (ii) comprises, a long-chain monobasicacid with a number of carbon atoms in a carbon chain greater than 9;(iii) comprises any one or more selected from the group consisting of amonobasic acid having 9 carbon atoms, a monobasic acid having 10 carbonatoms, a monobasic acid having 11 carbon atoms, a monobasic acid having12 carbon atoms, a monobasic acid having 13 carbon atoms, a monobasicacid having 14 carbon atoms, a monobasic acid having 15 carbon atoms, amonobasic acid having 16 carbon atoms, a monobasic acid having 17 carbonatoms, a monobasic acid having 18 carbon atoms, and a monobasic acidhaving 19 carbon atoms; or (iv) is decreased in content by at least 5%,at least 10%, at least 20%, at least 40%, at least 50% or more, comparedto that in the long-chain dibasic acid produced by fermentation with amicroorganism not containing the isolated DNA molecule of claim 1 I).